Use of a High-Throughput Phenotypic Screening Strategy to Identify Amplifiers, a Novel Pharmacological Class of Small Molecules That Exhibit Functional Synergy with Potentiators and Correctors.

Cystic fibrosis (CF) is a lethal genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite recent groundbreaking approval of genotype-specific small-molecule drugs, a significant portion of CF patients still lack effective therapeutic options that address the underlying cause of the disease. Through a phenotypic high-throughput screen of approximately 54,000 small molecules, we identified a novel class of CFTR modulators called amplifiers. The identified compound, the characteristics of which are represented here by PTI-CH, selectively increases the expression of immature CFTR protein across different CFTR mutations, including F508del-CFTR, by targeting the inefficiencies of early CFTR biosynthesis. PTI-CH also augments the activity of other CFTR modulators and was found to possess novel characteristics that distinguish it from CFTR potentiator and corrector moieties. The PTI-CH-mediated increase in F508del-CFTR did not elicit cytosolic or endoplasmic reticulum-associated cellular stress responses. Based on these data, amplifiers represent a promising new class of CFTR modulators for the treatment of CF that can be used synergistically with other CFTR modulators.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Modulation of the maladaptive stress response to manage diseases of protein folding.

Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer's disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure-function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR), through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease.

Multivariate analysis of the geochemistry and mineralogy of soils along two continental-scale transects in North America.

Soils collected in 2004 along two North American continental-scale transects were subjected to geochemical and mineralogical analyses. In previous interpretations of these analyses, data were expressed in weight percent and parts per million, and thus were subject to the effect of the constant-sum phenomenon. In a new approach to the data, this effect was removed by using centered log-ratio transformations to 'open' the mineralogical and geochemical arrays. Multivariate analyses, including principal component and linear discriminant analyses, of the centered log-ratio data reveal the effects of soil-forming processes, including soil parent material, weathering, and soil age, at the continental-scale of the data arrays that were not readily apparent in the more conventionally presented data. Linear discriminant analysis of the data arrays indicates that the majority of the soil samples collected along the transects can be more successfully classified with Level 1 ecological regional-scale classification by the soil geochemistry than soil mineralogy. A primary objective of this study is to discover and describe, in a parsimonious way, geochemical processes that are both independent and inter-dependent and manifested through compositional data including estimates of the elements and corresponding mineralogy.